Delta weight loss unlike genetic variation associates with hyperoxaluria after malabsorptive bariatric surgery

The risk of enteric hyperoxaluria is significantly increased after malabsorptive bariatric surgery (MBS). However, its underlying determinants are only poorly characterized. In this case–control study, we aimed at identifying clinical and genetic factors to dissect their individual contributions to the development of post-surgical hyperoxaluria. We determined the prevalence of hyperoxaluria and nephrolithiasis after MBS by 24-h urine samples and clinical questionnaires at our obesity center. Both hyperoxaluric and non-hyperoxaluric patients were screened for sequence variations in known and candidate genes implicated in hyperoxaluria (AGXT, GRHPR, HOGA1, SLC26A1, SLC26A6, SLC26A7) by targeted next generation sequencing (tNGS). The cohort comprised 67 patients, 49 females (73%) and 18 males (27%). While hyperoxaluria was found in 29 patients (43%), only one patient reported postprocedural nephrolithiasis within 41 months of follow-up. Upon tNGS, we did not find a difference regarding the burden of (rare) variants between hyperoxaluric and non-hyperoxaluric patients. However, patients with hyperoxaluria showed significantly greater weight loss accompanied by markers of intestinal malabsorption compared to non-hyperoxaluric controls. While enteric hyperoxaluria is very common after MBS, genetic variation of known hyperoxaluria genes contributes little to its pathogenesis. In contrast, the degree of postsurgical weight loss and levels of malabsorption parameters may allow for predicting the risk of enteric hyperoxaluria and consecutive kidney stone formation.

Ethical approval. The Leipzig Obesity BioBank (LOBB, https:// www. helmh oltz-munich. de/ en/ hi-mag/ cohort/ leipz ig-obesi ty-bio-bank-lobb) was approved by the Ethics committee of the University of Leipzig (Ethics vote 017-12-23012012). The study participants gave their written informed consent prior to tissue sampling and data collection. For patients not included in the LOBB, written informed consent was obtained before blood collection (Ethics vote 159/14-ff. University of Leipzig). All methods were performed in accordance with the relevant guidelines and regulations. All raw data used for statistical analysis is available in the Supplement.

Parameters
Postsurgical hyperoxaluria is related to reduced kidney function. Post-surgery kidney function was estimated by serum creatinine levels and eGFR at the individual follow-up time point and showed no significant differences between the patient groups HO and control (Table 1; Fig. 1BI ,CI). When dividing into subgroups, differences in creatinine remained insignificant (Table 1; Fig. 1BII). However, 'HO high' patients exhibited a reduced mean eGFR compared to 'HO moderate' patients (Table 1; Fig. 1CII). In addition, we observed a trend for higher urinary oxalate excretion values with increasing follow-up time post-surgery, which was, however, characterized by a remarkable variability at any time point (Fig. 1D). In line with the overall lower kidney function in the 'HO high' group, we detected a tendency for lower eGFR values in patients with higher oxalate excretion (Fig. 1E). Interestingly, when comparing pre-and postoperative mean eGFR values, we found a significant increase (p = 0.0014) within the control group but not in hyperoxaluric patients (p = 0.36) (Fig. 1F).

Impact of hyperoxaluria-associated genes in the development of postsurgical hyperoxaluria.
To evaluate the contribution of genetic factors in the development of hyperoxaluria after bariatric surgery, we conducted tNGS to detect variations within genes known to cause monogenic hyperoxaluria and Ca-Ox-NL (AGXT, GRHPR, HOGA1; SLC26A1) as well as the candidate genes SLC26A6 and SLC26A7 ( Fig. 2A). We hypothesized that MBS-associated enteric hyperoxaluria manifests upon genetic susceptibility, which is conveyed via hypomorphic variants in genes involved in hepatic, renal, and intestinal oxalate homeostasis. The mean coverage of tNGS was 477× for analyzed regions of interest. However, diagnostic validity was limited by an amplicon drop-out rate of 9%. Within the six genes analyzed, we identified 13 variants in 12 patients, 6 of which were hyperoxaluric (HO) and 6 non-hyperoxaluric (control) (HO: 1 × GRHPR, 5 × SLC26A1; control: Table S1). By analyzing individual oxalate excretion values over follow-up time, we found a trend for higher urinary oxalate excretion with increasing time in patients with genetic findings versus patients without findings (Fig. 2C). However, this result was of limited significance regarding the high variability at each time point and the low number of values at longer time intervals. In addition, the mean urinary oxalate level in variant carriers was not significantly increased in comparison to non-variant carriers (0.539 ± 0.09 mmol/day and 0.445 ± 0.030 mmol/day, respectively; p = 0.55) (Fig. 2D). Weight loss between both groups was slightly lower in patients with genetic findings (22.6 ± 3.5% versus 22.7 ± 1.6%; p = 0.97) (Fig. 2E). In summary, we found no evidence for an impact of variation in known or candidate hyperoxaluria-associated genes in the development of postsurgical hyperoxaluria in our cohort.  Fig. 3BII). To evaluate potential differences in the degree of enteric absorption we analyzed serum levels of total protein and zinc (Table 1) as well as albumin and iron (Table 2). Indeed, hyperoxaluric patients exhibit significantly lower total serum protein levels compared to controls (HO: 68.6 ± 0.7 g/L; control: 70.9 ± 0.7 g/L) (p = 0.02), indicating a higher degree of malabsorption (Table 1; Fig. 3CI), a trend that was also observed by comparing subgroups (Table 1; Fig. 3CII). Levels of zinc (Table 1) and albumin ( Table 2) were also lower in HO (p = 0.1 and p = 0.2 respectively). In contrast, comparison of lipid metabolism (LDL-C, HDL-C, and triglycerides) and serum calcium presented non-significant in-between groups ( Table 2). www.nature.com/scientificreports/ In addition to grouped analyses, individual oxalate levels were analyzed to test for correlations with EWL, total weight loss, and total serum protein. In contrast to EWL, for which only a positive trend could be observed (Fig. 3D), total weight loss significantly correlated with urinary oxalate levels in the total cohort (r = − 0.3; p = 0.02) (Fig. 3E). Accordingly, we observed a significant negative correlation between total serum protein and urinary oxalate levels for all patients (r = 0.30; p = 0.01) (Fig. 3F). Furthermore, a significant positive correlation was detected between weight loss and follow-up time for hyperoxaluric patients (r = 0.66; p = 0.0001), whereas no relationship could be observed for non-hyperoxaluric patients (r = − 1.00, p = 0.55) (Fig. 3G). To illustrate changes in weight loss pre-and postoperatively between HO and control patients, we analyzed the percentage of patients belonging to different BMI-groups (BMI: < 35; 36-39; > 40 kg/m 2 ) (Fig. 3H). Compared to controls, the HO group preoperatively included a higher percentage of patients within the highest BMI group (BMI > 40 kg/ m 2 : 90% vs 84%) and also a higher percentage within the lowest BMI group postoperatively (BMI: < 35 kg/m 2 : 66% vs 47%). Logistic regression aimed to assess the likelihood of developing hyperoxaluria with weight loss as a predictor variable. Log-likelihood ratio test revealed statistical significance for `HO-high` and 'total' (Fig. 3I) (p = 0.0251 and p = 0.0228, respectively). Altogether, our analyses indicate a strong connection between the degree of body weight reduction and enteric hyperoxaluria.

Discussion
Our study shows a high prevelance of hyperoxaluria in a cohort of patients that underwent malabsorptive bariatric surgery: 43% of our patients presented values higher than 0.45 mmol/day over a mean follow-up time of 46.6 ± 6.8. Those results are in line with other studies that measured oxalate excretion after bariatric surgery. Valezi et al. 14 compared pre-and 1-year postoperative values and reported a significant increase in urinary oxalate excretion after RYGB with hyperoxaluria found in more than half of all patients, compared to only 4% before the intervention. Similarly, in a study by Park et al. 15 hyperoxaluria increased from 11% preoperatively to 42% after surgery. Numerous studies reported also an increased risk of hyperoxaluric nephrolithiasis in postbariatric patients after malabsorptive procedures [15][16][17][18][19][20] .
Unexpectedly, only one patient (1.5%) in our cohort exhibited postoperative Calcium-Oxalate-nephrolithiasis. Time between surgical procedure and stone formation was 3.3 years (40 months). The low number of postoperative nephrolithiasis in our cohort may be explained by the relatively short observation period, as in 67% of patients the time between MBS and the last follow-up was less than 40 months. Secondly, the cohort shows a high variability within follow-up times (range 1-153 months). This leads to possibly missed stone events later in the postoperative period. To address this limitation, we extended the follow-up period for stone formation by abstracting medical records for another 12 months, however, no further stone event was recorded.
The observation that bariatric surgery has a positive effect on kidney function (creatinine-based eGFR) is in line with other studies 21,22 . Overall, values of eGFR and creatinine improved significantly (eGFR: p = 0.0013 and creatinine: p = 0.0003, respectively) within our cohort when comparing pre-and postoperative values. In 2016, Chang et al. 22 compared 985 severely obese patients undergoing bariatric surgery (mainly RYGB) with 985 matched controls with up to 9 years of follow-up. They reported a 58% risk reduction of eGFR decline and a 57% risk reduction in doubling of serum creatinine or end-stage kidney disease (ESKD) compared with matched non-surgery patients. Imam et al. 21 studied kidney related outcomes of CKD stage 3 and 4 patients after bariatric surgery matched with an obese control group. They reported that eGFR in the bariatric group was 9.84 mL/ min/1.73 m 2 higher than in controls at a median follow-up of 3 years.
Interestingly, improvement in kidney function between pre-and postoperative values (eGFR and creatinine) was only present in our non-hyperoxaluric control group (Fig. 1F). This could be related to the fact that oxalate is a toxic metabolite 23 and may exert negative effects on kidney and other organ function. In a recent study, higher 24-h urinary oxalate excretion was found to be a risk factor for chronic kidney disease (CKD) progression and ESKD in individuals with CKD stages 2-4 24 . Additionally, plasma oxalate is known to increase with decreasing eGFR 25 . An inverse correlation between plasma oxalate and eGFR was described in PH-patients even at early CKD stages (stages 1-3b) 26 . And lastly, high plasma oxalate levels were found to increase the risk of sudden cardiac death in patients on dialysis 27 .
Regarding factors that put patients at risk for developing of post-surgical hyperoxaluria, we aimed to characterize genetic susceptibility including risk-alleles or hypomorphic variants next to environmental factors. www.nature.com/scientificreports/ Although predisposing genetic factors could not be defined, genetic susceptibility conveyed through sequence variants in other candidate genes cannot be excluded by our study. The malabsorptive effect in certain bariatric procedures causing weight loss has often been discussed as the reason for enteric hyperoxaluria after MBS. Asplin 28 explains this phenomenon as the result of fat malabsorption in the small intestine. Normally, intraluminal diet calcium binds to oxalate, builds an insoluble precipitate and is excreted in the feces. In postsurgical patients with fat malabsorption, diet calcium binds to the increased amounts of intraluminal fatty acids instead of oxalate. The soluble free oxalate reaches the colon and is available for passive and paracellular intestinal absorption (Fig. 4). Furthermore, intraluminal bile salts and fatty acids can also increase the membrane permeability in the bowel and thus augment oxalate absorption 29 . Various studies reported that especially restrictive types of bariatric surgery (e.g. sleeve gastrectomy, gastric banding) were not associated with an increased risk for postoperative hyperoxaluria or kidney stones [30][31][32] . In a study by Moreland et al. from 2017 33 , hyperoxaluria after RYGB correlated with the degree of steatorrhea, which was not the case before surgery. This supports the notion that MBS-associated hyperoxaluria derives from intestinal fat malabsorption.
It has also been proposed, that the risk for hyperoxaluria and CaOx-nephrolithiasis increases with the degree of malabsorption in bariatric procedures. The first study to report hyperoxaluria after RYGB in 2005 18 , distinguished between 'standard RYGB' (procedure in our study population) and 'malabsorptive RYGB' . For the latter, length of the common channel was at 75-125 cm. Patients undergoing 'malabsorptive RYGB' showed a higher risk for hyperoxaluria and CaOx-nephrolithiasis. Lieske et al. corroborated those findings in 2015 20 , indicating that the degree of hyperoxaluria depends on the length of the remaining common channel and thus the amount of mucosa available for absorption.
In our study, patients with elevated oxalate excretion showed significantly greater total weight loss than controls. A significant positive correlation between weight loss and follow up time in hyperoxaluric patients supports this finding, whilst controls presented a negative correlation. The greater weight loss in hyperoxaluric patients could be explained through a more effective post-surgical malabsorption. To evaluate the state of malabsorption between the two groups, we determined serum levels of 'malabsorption parameters' (total protein, albumin, zinc and iron). We noticed significantly lower levels of serum protein in hyperoxaluric patients versus controls. This finding further points to a higher degree of malabsorption in hyperoxaluric patients.
EWL is a metric often used to determine the efficiency of bariatric surgery. To calculate EWL, three variables are necessary: pre-and postoperative weight and the patients' ideal body weight (IBW) 34 . Unlike total weight loss, however, mean EWL showed no significant differences between hyperoxaluric patients and controls. This indicates, that delta weight loss, independent from reaching the individuals IBW, is a risk factor for developing hyperoxaluria after bariatric surgery.
Limitations. This study has some limitations: first, the cohort was of moderate size due to the single center character limiting generalizability. Second, for determining the prevalence of hyperoxaluria in our cohort, a onetime 24 h urine collection was used to represent the patient´s urinary oxalate excretion. Hence, we cannot fully exclude false positives and false negatives. Furthermore, there was no report of pre-procedural urinary oxalate values allowing for exclusion of pre-existing hyperoxaluria as opposed to MBS-associated hyperoxaluria. Finally, the use of creatinine-based eGFR in the setting of bariatric surgery shall be interpreted with caution due to the post-surgical decrease of muscle and fat mass 22 . Figure 3. Association of hyperoxaluria with weight loss and enteric malabsorption. (AI-CI) Scatter plots showing mean ± SEM values of EWL, total weight loss and total serum protein in HO, control and total. Student's unpaired t-test was used for statistical analysis. (AII-CII) Scatter plots showing mean ± SEM values of EWL, total weight loss and total serum protein in 'HO-high' , 'HO-moderate' , control and total. Ordinary oneway ANOVA was used for statistical analysis. (D-F) Simple linear regression of urinary oxalate excretion with EWL, total weight loss and total serum protein, showing significant correlation for higher oxalate excretion with higher total weight loss and lower total serum protein (total cohort). (G) Simple linear regression of total weight loss and follow-up time showing significant correlation between weight loss and follow-up time for HO and total. (H) Spectrum of BMI-groups pre-and postoperatively between HO, control and total. (I) Simple logistic regression showing the likelihood of developing hyperoxaluria with weight loss as a predictor variable. Loglikelihood ratio test (LRT) was used for statistical analysis. *p < 0.05; BMI body mass index, EWL excess weight loss, HO hyperoxaluria, ns not significant. www.nature.com/scientificreports/

Conclusion
Our study demonstrates that hyperoxaluria is a common adverse event of malabsorptive bariatric surgery. We did not identify associated genetic determinants, rather the degree of postsurgical weight loss and levels of malabsorption serum parameters may allow for estimating the risk of enteric hyperoxaluria and consecutive kidney stone formation in the future. Urinary oxalate derives from two main sources: endogenous oxalate production in the liver as well as absorption of exogenous oxalate in the intestine. In primary hyperoxaluria (PH), an overproduction of oxalate in the liver results in hyperoxalemia and consecutively hyperoxaluria with possible kidney stone formation due to biallelic mutations in the genes AGXT, GRHPR, or HOGA1. These genes encode for peroxisomal or mitochondrial liver enzymes involved in pyruvate/glyoxylate metabolism. Malabsorptive bariatric surgery can cause enteric (secondary) hyperoxaluria. Enteric malabsorption leads to decreased absorption of intraluminal protein and fatty acids. Diet calcium binds to the increased amounts of intraluminal fatty acids instead of oxalate. Soluble free oxalate is now available for intestinal absorption leading to hyperoxalemia and hyperoxaluria.